Of all the energy sources available to mankind today, the most plentiful and probably most under-utilized is the energy from the sun that is converted by plants via photosynthesis and stored as a carbon source. (Demain et al., “Cellulase, Clostridia and Ethanol,” Micro Mol Biol Rev 69(1):124-154 (2005)). On a worldwide basis, terrestrial plants produce 1.3×103 metric tons (dry weight basis) of wood, which is equivalent to 7×109 metric tons of coal, or about two-thirds of the world's energy requirement. (Demain et al., “Cellulase, Clostridia and Ethanol,” Micro Mol Biol Rev 69(1):124-154 (2005)). Available cellulosic feedstocks from agriculture and other sources are about 180 million tons per year (Lynd, et al., Bioenergy: Background, Potential, and Policy, Senate Agricultural Hearings (2003)). Furthermore, tremendous amounts of cellulose are available as municipal and industrial wastes which today contribute to pollution problems. Thus, great interest exists in the use of cellulosic biomass as a renewable source of energy via breakdown to sugars that can then be converted to liquid fuel. (Demain et al., “Cellulase, Clostridia and Ethanol,” Micro Mol Biol Rev 69(1):124-154 (2005)).
The C. thermocellum cellulosome is a very large cellulase aggregate with a total molecular weight of millions and capable of degrading crystalline cellulose efficiently. Ever since the cellulosomal structure was discovered, it has been recognized that its activity is governed by its unique quaternary structure. The core of the cellulosome is a 250-kDa non-catalytic polypeptide, CipA, which binds to cellulose and serves as a scaffold for the catalytic subunits. CipA contains a series of nine highly homologous domains, termed the cohesin, which serve as receptors for the catalytic subunits. Binding to the cohesin domain is mediated by a highly conserved duplicated sequence of 22 amino acid residues, called the dockerin, which is mostly found at the C-terminus of each cellulosomal catalytic subunit. More than sixty subunits, mostly glycosyl hydrolases, have been found to contain the dockerin. These subunits include endoglucanases, exoglucanases, xylanases, and other hemicellulases. CipA further contains a cellulose-binding domain (CBD), which anchors the array of catalytic components to the cellulose surface.
A particularly attractive solution to the problem of excess waste and the need for alternative energy sources is the conversion of lignocellulosic biomass into motor fuel, i.e. ethanol, by a co-culture of thermophilic, anaerobic microorganisms, for example, a co-culture consisting of a cellulolytic strain such as C. thermocellum and a saccharolytic strain, such as C. thermosaccharolyticum. Together, these strains attack cellulose and hemicellulose and convert the sugars produced to ethanol. (Demain et al., “Cellulase, Clostridia and Ethanol,” Micro Mol Biol Rev 69(1):124-154 (2005)). Useful reviews on the biological conversion of lignocellulosic biomass to ethanol have been published. (Lee, J., “Biological Conversion of Lignocellulosic Biomass to Ethanol,” J Biotechnol 56:1-24 (1997); Lynd, L. R., “Large-Scale Fuel Ethanol From Lignocellulose. Potential, Economics, and Research Priorities,” App. Biochem Biotechnol 24, 25:695-719 (1990); Lynd, L. R., “Overview and Evaluation of Fuel Ethanol From Cellulosic Biomass: Technology, Economics, the Environment, and Policy,” Annu Rev Energy Environ 21:403-465 (1996); Lynd, L. R., “Production of Ethanol From Lignocellulosic Material Using Thermophilic Bacteria: Critical Evaluation of Potential and Review,” Adv Biochem Eng/Biotechnol 38:1-52 (1989); Lynd et al., “Fuel Ethanol from Cellulosic Biomass,” Science 251:1318-1323 (1991); Lynd et al., “Likely Features and Costs of Mature Biomass Ethanol Technology,” Appl Biochem Biotechnol 57/58:741-761 (1996); Lynd et al., “Microbial Cellulose Utilization: Fundamentals and Biotechnology,” Microbiol Mol Biol Rev 66:506-577 (2002); Lynd. et al., “Biocommodity Engineering,” Biotechnol Prog 15:777-793 (1999); Mielenz, J. R., “Ethanol Production From Biomass: Technology and Commercialization Status,” Curr Opin Microbiol 4:324-329 (2001); Wyman, C. E., “Potential Synergies and Challenges in Refining Cellulosic Biomass to Fuels, Chemicals, and Power,” Biotechnol Prog 19:254-262 (2003); Wyman et al., “Biotechnology for Production of Fuels, Chemicals, and Materials from Biomass,” Appl Biochem Biotechnol 39/40: 41-59 (1993); Wyman et al., “Ethanol Fundamentals of Production From Renewable Feedstocks and Use as a Transportation Fuel,” Appl Biochem Biotechnol 24/25:735-753 (1990).)
Attention has focused on anaerobic thermophiles as “ethanologens” for the following reasons: (i) thermophiles are thought to be robust and contain stable enzymes; (ii) anaerobes generally have a low cellular growth yield, hence more of the substrate is converted to ethanol; (iii) thermophilic fermentations are less prone to detrimental effects of contamination; (iv) growth at higher temperatures may facilitate the removal and recovery of volatile products such as ethanol. (Demain et al., “Cellulase, Clostridia and Ethanol,” Micro Mol Biol Rev 69(1):124-154 (2005)). Also extremely important are the advantages of cellulase production in situ and the high rates of metabolism of cellulose and hemicellulose.
In addition to addressing a pollution problem, the clostridial co-culture system is potentially capable of dramatically increasing the use of ethanol as a major liquid fuel using renewable photosynthetic biomass as feedstock. The major obstacle to an economic process is the production of the side-products, acetate and lactate, which limits conversion yield. In principle, the concept of a thermophilic ethanol fermentation is a very simple one involving a high-temperature fermentation with reduced need for power-consuming cooling and agitation/aeration of reactor vessels and with the four biologically-mediated events involved in ethanol production (cellulase and hemicellulase formation, cellulose and hemicellulose hydrolysis, hexose fermentation, and pentose fermentation) consolidated in a single process step. By combining recombinant DNA technology and metabolic engineering knowledge, drawbacks in the current methodologies of ethanol production may be overcome. However, most studies on the cellulosome have thus far focused on molecular cloning and characterization of the cellulosomal and non-cellulosomal enzymes, as well as the structure-function relationship of the proteins involved in biomass degradation. Little is known about how the biosynthesis of these proteins is regulated. The task of elucidating the regulatory mechanism is obviously complicated by the large number of the genes and proteins involved.
Cellulase synthesis is known to be controlled by transcription regulators. In the fungus Trichoderma reesei, a series of activators and repressors have been found to control the levels of cellulase and xylanase. ACEI serves as a repressor (Aro et al., “ACEI of Trichoderma reesei is a Repressor of Cellulase and Xylanase Expression,” Appl Environ Microbiol 69:56-65 (2003)) whereas ACEII (Aro et al., “ACEII, a Novel Transcriptional Activator Involved in Regulation of Cellulase and Xylanase Genes of Trichoderma reesei,” Biol Chem 276:24309-24314 (2001)) serves as an activator. In addition, CRE1 mediates glucose repression (Aro et al., “ACEI of Trichoderma reesei is a Repressor of Cellulase and Xylanase Expression,” Appl Environ Microbiol 69:56-65 (2003); Aro et al., “ACEII, a Novel Transcriptional Activator Involved in Regulation of Cellulase and Xylanase Genes of Trichoderma reesei,” Biol Chem 276:24309-24314 (2001); Saloheimo et al., “Carbohydrases From Trichoderma reesei and other Microorganisms,” Royal Society of Chemistry, Cambridge UK 267-279). The soil bacteria Thermobifida fusca (formerly Thermomonospora fusca) has six known cellulase genes, celA-celF. A protein that binds to a 14 bp inverted repeat found in the promoter region of each cellulase gene has been isolated (Spiridonov et al., “Characterization and Cloning of celR, a Transcriptional Regulator of Cellulase Genes from Thermomonospora fusca,” Biol Chem 274:13127-13132 (1999)). This protein, called CelR, serves as a repressor. Binding of CelR to its target DNA sequence is specifically inhibited by low concentrations of cellobiose (0.2-0.5 mM). A mutant of CelR with a slightly modified hinge helix protein structure has confirmed many of these results (Spiridonov et al., “A celR Mutation Affecting Transcription of Cellulase Genes in Thermobifida fusca,” J Bacteriol 182:252-255 (2000)). The mutation has been shown to cause weaker DNA binding than the wild type protein. CelR is constitutively expressed with posttranslational modifications affecting its DNA binding activity.
Unlike these microorganisms which produce only free cellulases, C. thermocellum produces the cellulosome in addition to free enzymes. A large number of the cellulosome components can be classified into three categories: 1) the scaffolding protein (CipA), 2) the dockerin-containing subunits (such as CelS and many others), and 3) the scaffoldin-anchorage proteins which anchor the cellulosome to the cell surface (such as OlpA, OlpB, and Orf2p). The second category alone comprises more than 60 different genes. The long list of the cellulosomal genes is further complicated by many non-cellulosomal cellulase components produced by this bacterium. The shear number of the genes involved in cellulose degradation suggests that regulation of cellulase biosynthesis in this bacterium is complicated.
Regulation of the cellulosomal cellulase and hemicellulase biosynthesis has been studied in the anaerobe, C. cellovorans (Han et al., “Regulation of Expression of Cellulosomal Cellulase and Hemicellulase Genes in Clostridium Cellulovorans,” Bacteriol 185:6067-6075 (2003)). The cellulosomal cellulase and hemicellulase genes are expressed into both monocistronic and polycistronic mRNAs. Transcription starts sites are found 61-233 bp upstream from the first nucleotide of each of the respective translation initiation codons. Some cellulase and hemicellulase genes in this bacterium are coordinately regulated by the carbon source present in the medium (Han et al., “Transcription of Clostridium Cellulovorans Cellulosomal Cellulase and Hemicellulase Genes,” Bacteriol 185:2520-2527 (2003)). Furthermore, a catabolite repression type of mechanism regulates cellulase expression.
In C. thermocellum, regulation of CelS, the major component of the cellulosome, has been studied at the protein level using western blot. The results indicate that CelS production is higher on cellulose than cellobiose (Dror et al., “Regulation of the Cellulosomal CelS (cel48A) Gene of Clostridium thermocellum is Growth Rate Dependent,” Bacteriol 185:3042-3048 (2003)). Quantitative RNase protection assay revealed that the level of celS mRNA under carbon or nitrogen limitation in a chemostat is a function of the growth rate, lower rate favoring celS expression. Two major transcriptional start sites are found 145 and 140 bp upstream of the translational start site, respectively. The relative activities of the two promoters remain constant under the expression conditions. Similar experiments have been done with the scaffoldin-related genes of the bacterium (Dror et al., “Regulation of Expression of Scaffoldin-Related Genes in Clostridium thermocellum,” Bacteriol 185:5109-5116 (2003)). The transcription levels of cipA, olpB, and orf2A vary with the growth rate under nitrogen or carbon limitation. On the other hand, expression of sdbA is independent from the growth rate. Two transcription start sites have been found 81 and 50 bp upstream of the CipA translational start site, respectively. Transcription from the first promoter (σL-like) occurs under all growth conditions, whereas expression from the second promoter (sA-like) occurs only under carbon limitation.
Identification and characterization of transcription regulators is an important step in understanding the control of cellulase biosynthesis in bacterium, however, no regulators of cellulase synthesis are heretofore identified. What is needed now is identification and characterization of specific transcription regulators of cellulose and hemicellulose synthesis by thermophilic anaerobic microorganisms. Armed with an understanding of transcription regulation of cellulase and hemicellulase synthesis by anaerobes such as Clostridium spp., recombinant technology can be partnered with metabolic engineering techniques to develop practical and far-reaching solutions to the problems of excess cellulosic waste and the need for alternative energy sources through the efficient conversion of cellulosic biomass to ethanol.
The present invention is directed to overcoming these and other deficiencies in the art.